The major focus of this project is to investigate the molecular biology and biochemistry of the ras oncogenes and the p21 ras oncogene proteins. the long-range goal is to elicidate molecular mechanisms of cell transformation induced by these genes and their protein products. We have characterized in detail the biochemical properties of p21 overproduced in E. eoli. The GTP/GDP binding, autokinase and GTPase activities of this fusion protein are very similar to p 21 in mammalian cells. Importantly, we have found that these p 21 activities can be specifically inhibited by a monoclonal antibody (Y13-259), indicating that all these activities are conferred by a single active center within the p21 molecule. This observation, together with the finding by Stacey et al. that the same monoclonal antibody can specifically block p21 in vivo function, provides the first direct evidence to implicate the p21 GTP/GDP binding in its cellular function. The active center of p21 has also been probed by photoaffinity labeling and other chemical methods. Extensive studies are undertaken to delineate the structural requirement of p21 for its biological activity by construction of many p21 mutant proteins with specific amino acid substitutions. The p21 post-translation acylation site has been mapped by a synthetic tetrapeptide of the p21 C-terminus. p21 is palmitylated at cysteine-196, presumably through a thieoster linkage. The fatty acid is identified as palmitic acid, not myristic acid as has been observed in N-terminal acylation of oncogene proteins such as p60 src. The functional relationship of p21 to G proteins of adenylate cyclase has been evaluated, and attempts are being made to identify p21 cellular targets by chemical cross-linking.